Abstract
Visualization of the herpesvirus genomes during lytic replication and latency is mainly achieved by fluorescence in situ hybridization (FISH). Unfortunately, this technique cannot be used for the real-time detection of viral genome in living cells. To facilitate the visualization of the Marek’s disease virus (MDV) genome during all stages of the virus lifecycle, we took advantage of the well-established tetracycline operator/repressor (TetO/TetR) system. This system consists of a fluorescently labeled TetR (TetR-GFP) that specifically binds to an array of tetO sequences. This tetO repeat array was first inserted into the MDV genome (vTetO). Subsequently, we fused TetR-GFP via a P2a self-cleaving peptide to the C-terminus of the viral interleukin 8 (vIL8), which is expressed during lytic replication and latency. Upon reconstitution of this vTetO-TetR virus, fluorescently labeled replication compartments were detected in the nucleus during lytic replication. After validating the specificity of the observed signal, we used the system to visualize the genesis and mobility of the viral replication compartments. In addition, we assessed the infection of nuclei in syncytia as well as lytic replication and latency in T cells. Taken together, we established a system allowing us to track the MDV genome in living cells that can be applied to many other DNA viruses.
Highlights
Marek’s disease virus (MDV), known as Gallid alphaherpesvirus 2 (GaHV-2), is a highly oncogenic herpesvirus that belongs to the genus Mardivirus
The TetR fused to a fluorescence protein and a nuclear localization signal (NLS) and was either expressed by the virus or the cells depending on the application
To exclude that the TetR protein alone forms unspecific aggregates in the nucleus of chicken cells, we generated a virus expressing high levels of TetR-green fluorescent protein (GFP) driven by the strong thymidine kinase (TK) promoter in the mini-F in the absence of tetO
Summary
Marek’s disease virus (MDV), known as Gallid alphaherpesvirus 2 (GaHV-2), is a highly oncogenic herpesvirus that belongs to the genus Mardivirus. The virus enters the host through the respiratory tract where it infects macrophages and dendritic cells [2,3] that transport the virus to lymphoid organs. This cell-associated virus is passed on to B and T cells in which it can replicate lytically [4,5,6]. MDV establishes latency predominantly in CD4+ T cells [7,8] and integrates its genome into the host telomeres [9,10]. Only a few genes are expressed, including the major oncogene Meq (MDV005 and MDV076), splice variants of the viral chemokine vCXCL13
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